CN104095883A - Coral extract, its extraction method and use, and coral extract health product - Google Patents
Coral extract, its extraction method and use, and coral extract health product Download PDFInfo
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- CN104095883A CN104095883A CN201410066589.5A CN201410066589A CN104095883A CN 104095883 A CN104095883 A CN 104095883A CN 201410066589 A CN201410066589 A CN 201410066589A CN 104095883 A CN104095883 A CN 104095883A
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- corallium japonicum
- japonicum kishinouye
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention discloses a coral extract for skin whitening, moisturizing, elasticity improving, anti-inflammation and wound healing and an extraction method thereof; the coral extract is an extract of Corallium japonicum Kishinouye (Briareumexcavatum); the extraction method comprises the following steps: extracting with organic solvent, extracting with water and ethyl acetate, and separating by column chromatography; the coral extract can be used in the fields of skin whitening, moisture retention, elasticity improvement, inflammation resistance, wound healing and the like.
Description
Technical field
The present invention relates to a kind of Corallium Japonicum Kishinouye extract, extracting process and purposes, refer in particular to a kind of soft and soggy Corallium Japonicum Kishinouye (Briareum excavatum), this extract has the skin moisture-keeping of raising, elasticity, inhibition Tyrosinase activity, reduces melanin formation, and slows down inflammatory cells infiltration and control the effects such as wound area.
Background technology
The biosynthesis pathway of secondary metabolite that marine organisms is produced due to the particularity of the marine eco-environment has significant difference compared with terrestrial life with enzymatic reaction system, cause marine organisms often can produce various the having and the ocean secondary metabolite of the complete different structure feature of terrestrial life of some chemical constitution novelties, biological activity.
Corallium Japonicum Kishinouye belongs to Coelenterata, has more than 9000 to plant, and is one of modal biology in ocean, accounts for 22.4% of marine organisms kind, is the living marine resources that can utilize in a large number.Soft coral order (gorgonian) is commonly called as cockle, extra large whip, Hai Liu, is the large branch of one in Corallium Japonicum Kishinouye animal.In to the research of Corallium Japonicum Kishinouye chemical composition, since the Weinheiner of the U.S. equals within 1969, to find the abundant prostaglandin precursor with unique texture and strong physiologically active from soft coral order, attract numerous natural product chemistry men that object of study is turned to marine organisms from terrestrial life, a large amount of found marine natural productss have biological activity widely, Main Function is in nervous system, cardiovascular system, immune system etc., and manyly has a significant anti-tumor activity.
Still very active to the research of soft coral in recent years, for example, the soft coral order belonging to from Briareum, find numerous activated briarane type diterpene, but be limited to the restriction in wild Corallium Japonicum Kishinouye source, therefore there is scholar to attempt the soft and soggy Corallium Japonicum Kishinouye in soft coral order (Briareum excavatum) to carry out laboratory artificial cultivating, but case once disclosed before there is no so far any data in literature or patent, can obtain having from soft and soggy Corallium Japonicum Kishinouye and suppress Tyrosinase activity, accelerating wound healing extraction product that can anti-inflammatory or purified compound.
Summary of the invention
The object of the present invention is to provide a kind of Corallium Japonicum Kishinouye extract, it is obtained by soft and soggy Corallium Japonicum Kishinouye (Briareum excavatum) extraction, there is the skin moisture-keeping of raising, elasticity, inhibition Tyrosinase activity, reduce melanin formation, and slow down inflammatory cells infiltration and control the effects such as wound area.
Another object of the present invention is to provide a kind of extracting process of Corallium Japonicum Kishinouye extract, the easy volume production that is easy to of its method.
A further object of the present invention is to provide a kind of purposes of Corallium Japonicum Kishinouye extract, and it is applied to aforesaid Corallium Japonicum Kishinouye extract the skin of one live body, so that skin-whitening, moisturizing, improve skin elasticity, anti-inflammatory and can impel wound healing.
In order to reach above-mentioned purpose, solution of the present invention is:
For skin-whitening, moisturizing, improve the Corallium Japonicum Kishinouye extract of elasticity, anti-inflammatory and wound healing, it is obtained by the following step:
A. with dry soft and soggy Corallium Japonicum Kishinouye (Briareum excavatum) sample of an organic liquid mixture extraction, obtain just extract of organic layer, the C1-C4 alcohols that wherein this organic liquid mixture comprises equal proportion and the chloride alkanes of C1-C4; B. distribute just extract of this organic layer of extraction with water and rudimentary esters, obtain a rudimentary esters and slightly extract thing, this rudimentary esters slightly extracts thing and contains a Corallium Japonicum Kishinouye active component of 5～50%.
This rudimentary esters of above-mentioned b. step slightly extracts thing further with tubing string chromatographic analysis purification in addition, it rushes extract punching with one and carries this rudimentary esters and slightly extract thing, with one rush extract, wherein this rushes rushing of extract and puies forward gradient and provided by one first mixed solvent of n-hexane/ethyl acetate and one second mixed solvent of ethyl acetate/methanol.
The C1-C4 alcohols of above-mentioned a. step is methanol.
The chloride alkanes of the C1-C4 of above-mentioned a. step is dichloromethane.
The rudimentary esters of above-mentioned b. step is ethyl acetate.
In above-mentioned the first mixed solvent, the solvent gradient of normal hexane is 0%-99%.
In above-mentioned the second mixed solvent, the solvent gradient ratio of ethyl acetate is 0%-90%.
Above-mentioned Corallium Japonicum Kishinouye active component slightly extracts thing by this rudimentary esters and is further rushed and carried and obtain by this first mixed solvent, and wherein rushing of this first mixed solvent put forward gradient and be: normal hexane: the division (fraction) that ethyl acetate is 90:10 to 70:30.
It is 5-30% that above-mentioned Corallium Japonicum Kishinouye active component slightly extracts thing proportion in this rudimentary esters after punching is carried.
Above-mentioned Corallium Japonicum Kishinouye active component slightly extracts thing by this rudimentary esters and is further rushed and carried and obtain by this first mixed solvent, and wherein rushing of this first mixed solvent put forward gradient and be: normal hexane: the division (fraction) that ethyl acetate is 60:40 to 50:50.
It is 20-50% that above-mentioned Corallium Japonicum Kishinouye active component slightly extracts thing proportion in this rudimentary esters after punching is carried.
An extracting process for described Corallium Japonicum Kishinouye extract, comprises:
A. with dry soft and soggy Corallium Japonicum Kishinouye (Briareum excavatum) sample of an organic liquid mixture extraction, obtain just extract of organic layer, the C1-C4 alcohols that wherein this organic liquid mixture comprises equal proportion and the chloride alkanes of C1-C4; B. distribute just extract of this organic layer of extraction with water and rudimentary esters, obtain a rudimentary esters and slightly extract thing, this rudimentary esters slightly extracts thing and contains a Corallium Japonicum Kishinouye active component of 5～50%.
This rudimentary esters of above-mentioned b. step slightly extracts thing further with tubing string chromatographic analysis purification in addition, it rushes extract punching with one and carries this rudimentary esters and slightly extract thing, with one rush extract, wherein this rushes rushing of extract and puies forward gradient and provided by one first mixed solvent of n-hexane/ethyl acetate and one second mixed solvent of ethyl acetate/methanol.
A purposes for Corallium Japonicum Kishinouye extract, the Corallium Japonicum Kishinouye extract described in it is applied to the skin of a live body, so that skin-whitening.
A purposes for Corallium Japonicum Kishinouye extract, it is applied to described Corallium Japonicum Kishinouye extract the skin of one live body, so that skin moisture-keeping.
A purposes for Corallium Japonicum Kishinouye extract, it is applied to described Corallium Japonicum Kishinouye extract the skin of one live body, to improve skin elasticity.
A purposes for Corallium Japonicum Kishinouye extract, it uses described Corallium Japonicum Kishinouye extract, with anti-inflammatory.
A purposes for Corallium Japonicum Kishinouye extract, it uses described Corallium Japonicum Kishinouye extract, to impel wound healing.
A kind of skin care products, it comprises described Corallium Japonicum Kishinouye extract, the weight ratio of this Corallium Japonicum Kishinouye extract tool 0.00001～10%.
Adopt after said structure, the present invention is Corallium Japonicum Kishinouye extract and extracting process thereof, this Corallium Japonicum Kishinouye extract is obtained by the following step: a. is with dry soft and soggy Corallium Japonicum Kishinouye (Briareum excavatum) sample of an organic mixed extractant solvent, obtain just extract of organic layer, the C1-C4 alcohols that wherein this organic liquid mixture comprises equal proportion and the chloride alkanes of C1-C4; B. distribute just extract of this organic layer of extraction with water and rudimentary esters, obtain a rudimentary esters and slightly extract thing, this rudimentary esters slightly extracts thing and contains a Corallium Japonicum Kishinouye active component of 5～50%; It is obtained by soft and soggy Corallium Japonicum Kishinouye (Briareum excavatum) extraction, has the skin moisture-keeping of raising, elasticity, inhibition Tyrosinase activity, reduces melanin formation, and slow down inflammatory cells infiltration and control the effects such as wound area.
The purposes of Corallium Japonicum Kishinouye extract of the present invention, it is applied to aforesaid Corallium Japonicum Kishinouye extract the skin of one live body, so that skin-whitening, skin moisture-keeping, improve skin elasticity, anti-inflammatory and can impel wound healing.
Skin care products of the present invention comprises aforesaid Corallium Japonicum Kishinouye extract, and wherein Corallium Japonicum Kishinouye extract can be had a weight ratio of 0.00001～10%.
Brief description of the drawings
Fig. 1 is thick extraction and the separating step of the soft and soggy Corallium Japonicum Kishinouye of breeding type (Briareum excavatum) effective ingredient;
Fig. 2 is the 1H-NMR collection of illustrative plates of BP1;
Fig. 3 is the 1H-NMR collection of illustrative plates of BP2;
Fig. 4 is the 1H-NMR collection of illustrative plates of BP3;
Fig. 5 is the 1H-NMR collection of illustrative plates of BP4;
Fig. 6 is the 1H-NMR collection of illustrative plates of BP5;
Fig. 7 is the 1H-NMR collection of illustrative plates of BP6;
Fig. 8 is the 1H-NMR collection of illustrative plates of BP7;
Fig. 9 is that animal skin scratch test result is carried out in the each thick extraction of soft and soggy Corallium Japonicum Kishinouye;
Figure 10 is the experimental result of in vitro Tyrosinase active testing;
Figure 11 is the experimental result of in vitro melanin content test;
Figure 12 is the experimental result of BP2 live body whitening activity analysis;
Figure 13 is the experimental result of BP3 live body whitening activity analysis;
Figure 14 is the result that the fibroblast with mouse: NIH/3T3 carries out in vitro wound healing test experiments, and in figure, A, B are control group, do not give tester; C, D are BP2 low dose group, and concentration is 10 μ g/ml; E, F are BP2 high dose group, and concentration is 20 μ g/ml; G, H are BP3 low dose group, and concentration is 10 μ g/ml; I, J are BP3 high dose group, and concentration is 20 μ g/ml; K is the statistical data of each group;
Figure 15 is the result of carrying out in vitro wound healing test experiments with human umbilical vein endothelial cell's strain: EA.hy926, and in figure, A, B are control group, do not give tester; C, D are BP2 low dose group, and concentration is 10 μ g/ml; E, F are BP2 high dose group, and concentration is 20 μ g/ml; G, H are BP3 low dose group, and concentration is 10 μ g/ml; I, J are BP3 high dose group, and concentration is 20 μ g/ml; K is the statistical data of each group;
Figure 16 is the experimental result with the wound healing test of the BP2 of 3mg/0.2ml, BP3, the treatment of pure emulsion;
Figure 17 is with the BP2 of 40 μ g/0.2ml, BP3, slightly extracts the experimental result of the wound healing test of thing, pure emulsion treatment;
Figure 18 is the rat burn and scald skin histopathology section that utilizes HE dyeing (5X), and in figure, A is that control group, B are that burn and scald group, C are that BP2 group, D are BP3 group, gives natural goods concentration and is all 3mg/0.2ml;
Figure 19 is the rat burn and scald skin histopathology section that utilizes HE dyeing (5X), and in figure, A is that control group, B are that burn and scald group, C are that BP2 group, D are that BP3 group, E are thick extraction thing group, gives natural goods concentration and is all 40 μ g/0.2ml;
Figure 20 is that 3 kinds of embryo skin care productss and blank group and cream substrate are in moisturizing comparison;
Figure 21 is the comparison at elastic force of 3 kinds of embryo skin care productss and blank group and cream substrate.
Detailed description of the invention
In order further to explain technical scheme of the present invention, below by specific embodiment, the present invention will be described in detail.
Preparation example 1: with the soft and soggy Corallium Japonicum Kishinouye of organic solvent extraction
To cultivate in a large number in the soft and soggy Corallium Japonicum Kishinouye (1021.49g of biological sample Briareum excavatum of 0.6 ton of cultivation water vat, weight in wet base) carry out lyophilization, and dry Corallium Japonicum Kishinouye tissue is ground to (dry weight 417.75g), then at room temperature extract with organic solvent ethanol/methylene (1:1) mixed proportion, present after clarification through the organic solvent that repeatedly re-extract extremely adds, obtain just extract of organic layer.After more first extract being filtered, carry out concentrating under reduced pressure, the thick extraction thing obtaining distributes extraction with water and ethyl acetate, obtains the thick extraction thing (15.75g) of ethyl acetate layer after over-allocation extraction and concentrating under reduced pressure.Carry out again can obtaining after following separation process BP2 (2.44g, account for thick extraction thing 15.5%) and BP3 (5.6g, account for thick extraction thing 35.6%).
Preparation example 2: slightly extract thing separation process
Referring to Fig. 1, utilize tubing string chromatography to carry out preliminary separation to ethyl acetate layer, the filler of selecting is silica gel (Merck, 230-400mesh), do gradient punching and carry for rushing extract with the mixed solvent of n-hexane/ethyl acetate and ethyl acetate/methanol, be divided into seven divisions (fraction), each divide to rush the condition of putting forward as follows:
Divide 1 (BP1): Hexane:EtOAC (normal hexane: ethyl acetate) 99:1～93:7.
Divide 2 (BP2): Hexane:EtOAC (normal hexane: ethyl acetate) 90:10～70:30.
Divide 3 (BP3): Hexane:EtOAC (normal hexane: ethyl acetate) 60:40～50:50.
Divide 4 (BP4): Hexane:EtOAC (normal hexane: ethyl acetate) 40:60～20:80.
Divide 5 (BP5): Hexane:EtOAC (normal hexane: ethyl acetate) 10:90～0:100.
Divide 6 (BP6): EtOAC:MeOH (ethyl acetate: methanol) 90:10～50:50.
Divide 7 (BP7): EtOAC:MeOH (ethyl acetate: methanol) 40:60～0:100.
Each division afterwards detects with nuclear magnetic resonance analyser after concentrating under reduced pressure, and the signal (referring to Fig. 2 to Fig. 8) that obtains 1H-NMR collection of illustrative plates exists foundation as judge index composition, in order to carrying out the further foundation of biological activity test.
Test case 1: in vitro anti-inflammatory active testing
1. natural goods screening and cell strain use
Carry thick extract after step for gradient punching, use polysaccharide ester to bring out the in vitro inflammation pattern of mouse macrophage RAW264.7 cell strain and carry out screening operation.Control the mouse macrophage RAW264.7 number of 6 centimeters of culture dishs at 3x10
6individual, first give thick extract, then give ester polysaccharide (LPS) collecting cell after 16-18 hour.
2. west point method of the use of ink and water protein performance component analysis
Use 4% phosphate buffered solution (phosphate buffered saline, PBS) (137mM NaCl, 2.68mM KCl, 10mM Na
2hPO
4, 1.76mM KH
2pO
4, pH=7.2) be collected in 1.5ml centrifuge tube, after centrifugal 8 minutes with 3000rpm, remove supernatant, add again lysis buffer200 μ l (the 50mM Tris of 4 DEG C of temperature, pH7.5,150mM NaCl, 1%TritonX-100,0.1mM EDTA, 0.1mM EGTA, 100 μ g/ml phenylmethylsulfonyl fluoride, 1 μ g/ml Aprotinin, 20mM NaF, 0.2mM Na
3vO
4) break after cell membrane, under 4 DEG C of temperature, with 14,000rpm centrifugal 30 minutes, take out supernatant and also copy the people's such as Lowry method (Lowry et al., 1951) to carry out quantification of protein.
Use Bio-Rad DC protein assay kit (Hercules, CA, USA) and enzyme plate reading (Thermo Electron Corporation, USA) to analyze the light absorption value of supernatant, to measure the albumen quality of each specimen.By specimen buffer (the sample buffer of 1/3rd volumes of calibrated rear this cumulative volume of sampling; 2%SDS, 10%glycerol, 0.1%bromophenol bule, 10%2-mercaptoethanol, 50mM Tris, Bio-Rad Laboratories, inc).Utilize 7%, 10% SDS-PAGE with 80 volts of voltage isolated proteins.By the protein on SDS-PAGE with 135 milliamperes of electric current transposition overnight to pvdf membrane (0.45mm pore size, Immobilon-P, Millipore, Bedford, MA, USA).
Pvdf membrane after transfer printing is to take off ester milk powder TTBS solution (Tris-Tween buffer saline) (Tris-HCl20mM containing 5%, NaCl137mM, pH7.4,0.1%Tween20) at room temperature cover 40 minutes, more at room temperature react two hours with the one-level antibody for bringing out type nitric oxide synthetase and the effect of Second-Type Cycloxygenase of 1:1000 dilution ratio.Clean three times with TTBS solution subsequently, then react at room temperature one hour 30 minutes with the anti-rabbit IgG antibody (1:2000) of HRP-conjugated, after finishing, secondary antibody cleans three times with TTBS solution, finally utilize colour generation liquid (Immobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA01821U.S.A.) react with pvdf membrane, and with image analysing computer treatment facility (UVP Biospectrum AC System, UVP Inc, U.S.A.) detecting cold light reaction, the performance amount of record protein, with Computer aided analysis (VisionWorks LS Acquisition and analysis software, copyright2007, LLC, U.S.A.) detect and calculate relative quantity.And to add separately ester polysaccharide group as 100%, finally using α-actin as interior control group.
Its result compares the thick extract of different demarcation layer in the achievement of in vitro anti-inflammatory, is further used as experiment made on the living pattern and other isolated experiment patterns as reference.
3. the target antibody using
(1) bring out type nitric oxide synthetase (BD Pharmingen, San Diego, CA, USA:catalog no.6103322:polyclonal antibody), dilution ratio 1:1000.
(2) Second-Type Cycloxygenase (Cayman Chemical, USA; Catalog no.160106; Polyclonal antibody), dilution ratio 1:1000.
(3) β-actin (sigma, St.Louis, MO, USA; Catalog no.A5316-2ML; Monoclonal antibody), dilution ratio 1:2000.
4. result of the test
Referring to Fig. 9, it compares the thick extract of different demarcation layer in the effect of in vitro anti-inflammatory, found that the BP2 (dividing 2) of 20 μ g/ml and BP3 (dividing 3) have very significantly anti-inflammatory effect in vitro.
Test case 2: the in vitro Tyrosinase active testing of whitening activity analysis A. (in vitro tyrosinase activity assay)
1. test procedure
Test procedure is with reference to the research report of Wang (2010), and its entirety is all drawn the reference data as this case.B16-F10 kind is entered in the hole of 24 little lattice culture plates (24well plate), every little lattice kind enters 1*10
5individual cell, is positioned over 37 DEG C, 5%CO
2incubator after 24 hours, original cell culture fluid is removed, and adds the cell culture fluid that contains thick extraction thing, then put into incubator and cultivate 48 hours.After finishing, administration removes original cell culture fluid, and add 200 μ l PBS rinses once, then the 100 μ l trypsin effects that add make cell detachment bottom surface for 1～2 minute, then add in 200 μ l cell culture fluids and trypsin reaction, and celliferous culture fluid is packed in microcentrifugal tube and after centrifugal 5 minutes, removes supernatant with 3000rpm, add 200 μ l PBS rinses once, with 3000rpm centrifugal 5 minutes again, remove supernatant, add 100 μ l1%triton X-100/PBS, mix homogeneously makes cell breakage.Then at 4 DEG C with 10000rpm centrifugal 10 minutes, get supernatant and carry out quantification of protein.Protein under same concentration, gets equal-volume supernatant and L-dopa (2mg/ml) and mixes, and reacts the light absorption value that utilizes enzyme plate reading to survey wavelength 475nm after a hour and does sorting-out in statistics.
2. result of the test
Referring to Figure 10, its utilization adds variable concentrations BP2 and BP3 in melanoma cell, after 48 hours, carry out the experimental result of Tyrosinase activity analysis, wherein X-axis is each concentration medicine, Y-axis is in vitro Tyrosinase activity/melanin content, if the product producing the more, represent that catalysis intensity measures more by force.Found that thus under same protein concentration, the group that gives BP2 and BP3 all has the activity that suppresses Tyrosinase, and along with concentration promotes the trend that presents dosage interdependent (dose-dependent).
B. in vitro melanin content test (in vitro melanin content assay)
1. test procedure
Test procedure is with reference to the research report of Wang (2010), and its entirety is all drawn the reference data as this case.B16-F10 kind is entered in the hole of 24 little lattice culture plates, every little lattice kind enters 1*10
5individual cell, is positioned over 37 DEG C, 5%CO
2incubator after 24 hours, original cell culture fluid is removed, add the cell culture fluid that contains thick extraction thing, putting into incubator cultivated after 48 hours again, with 200 μ l PBS rinses once, the 100 μ l trypsin effects that then add try to get to the heart of a matter cell detachment in 1～2 minute, then add in 200 μ l cell culture fluids and trypsin reaction, pack in microcentrifugal tube with 3000rpm centrifugal 5 minutes into, remove supernatant.Add again 200 μ l PBS rinses once, and with 3000rpm centrifugal 5 minutes, remove after supernatant, add 100 μ l1N NaOH at 80 DEG C, to react one hour.The light absorption value that utilizes enzyme plate reading to survey wavelength 405nm does sorting-out in statistics.
2. result of the test
Referring to Figure 11, its utilization adds variable concentrations BP2 and BP3 after 48 hours, to carry out the experimental result of melanin (melanin) assay in melanoma cell.Cell is added after sodium hydroxide heating for dissolving, and while utilizing the absorption value that spectrophotometric determination wavelength is 490nm, absorption value is higher illustrates that melanin content is also higher.Matched by data show result and Tyrosinase activity, along with drug level is higher, the Tyrosinase activity of reduction is more remarkable, and melanic performance amount is also along with decline.
C. mushroom Tyrosinase suppresses capability analysis
1. experimental procedure
In 96well plate, in each hole, add the PB of 70 μ l, the inspection product solution of the various variable concentrations of 20 μ l, mix homogeneously.Then add the 12U Tyrosinase of 10 μ l, put into 37 DEG C of incubators and react 30 minutes.Add again the 15mM L-dopa of 10 μ l, continue to put into incubator and react after 30 minutes, under wavelength 492nm, survey the variable quantity of its light absorption value.With H
2o or 50%EtOH are as control group.Try to achieve the antioxygen rate of determinand with the restraint of tyrosinase percentage rate of following formula.Suppress lipid peroxy rate (%) higher, represent that non-oxidizability is stronger.
Restraint of tyrosinase percentage rate (Inhibition of Tyrosinase%, IT%)=[1-(sample is in the light absorption value of 492nm)/(not adding the control group of sample in the light absorption value of 492nm)] × 100.
2. experimental result
Tyrosinase is the speed factor of determination that melanin forms, if determinand can suppress the activity of Tyrosinase, just can reduce the generation of dopaquinone compounds, light absorption value measured under the wavelength of 492nm reduces relatively, can extrapolate by the height of light absorption value the ability that determinand suppresses Tyrosinase activity, and then can generate by check melanin.Experimental result discovery, has remarkable inhibition Tyrosinase activity at BP2 and the BP3 of 500 μ g/ml and 1000 μ g/ml, and its experimental result can see table 1:
D. live body whitening activity analysis
1. experimental procedure
A. planting fish raises:
Experiment is used four months above Brachydanio rerio (AB strain Danio rerio) of the age of a fish for experiment kind of fish, raises in having the acryl water vat of filter and blood circulation, and water temperature is controlled in 28.5 DEG C.It is 14 and 10 hours that light dark period is controlled respectively.
B. compounding medicine:
The medicine that wish detects must first be dissolved in 100%DMSO solution, and ultimate density must be considered DMSO and drug concentration simultaneously.
C. medicine gives:
Get the after fertilization embryo of 9 hours, inject 96 little lattice culture plates, in every little lattice, have Hank ' the s buffer of 3 embryos and 100 μ l, then the medicine preparing is got to 100 μ l and inject hole, fully on mix homogeneously bonnet, porose disc lid avoids moisture loss to cause concentration to change.Insert low temperature and irradiance incubator (Model RI-80, Firstek, Taiwan), control light dark period be respectively 14 with 10 hours (must be identical with the photoperiod of kind of fish), and temperature is maintained at 28.5 DEG C, continues immersion administration 48 hours.
D. fish body image capture
Put in poison latter 48 hours (being after fertilization 57 hours), take out juvenile fish with anesthetis (MS-222, 168ppm Tricaine) anesthesia after be sequentially positioned over individually concave slide (Micro Scientific Laboratories, Inc., U.S.A.) in groove, utilize 1% methyl fiber (Methyl Cellulose) (Sigma, U.S.A.) help fixing fish body recycling stero microscope (Z16APO, Leica, Heerbrugg, Switzerland) observe, collocation image acquisition system (idea SPOT, Diagnostic instruments Inc., and control software (SPOT software VERSION4.6 U.S.A.), Diagnostic instruments Inc., U.S.A.), obtain fish body image.This experiment time of exposure is made as 3.372msec.
E. date processing:
The fish body image of obtaining is utilized to image processing software (Image J1.43g; National Institute of Health, Bethesda, MD, USA) open, be made as 0 at lowest threshold, high threshold is made as under 85 scope, and the melanin value of Brachydanio rerio is respectively organized in quantitative analysis.
2. experimental result:
Referring to Figure 12 and Figure 13, observe the melanin value of Brachydanio rerio, surplus 50% left and right of Arbutin pigment content when 20mM (approximating 5446 μ g/ml), and along with the concentration of BP2 and BP3 is higher, pigment content drops to 70% gradually, although this experimental result BP2 and BP3 whitening degree do not have calibration control group superior, doubly, therefore these two kinds thick extraction things have the effective ingredient of splendid whitening potentiality to one of five percentages that its concentration is Arbutin.
Test case 3: wound healing promoting activity analysis
A. wound healing promoting activity analysis
1. experimental procedure
With fibroblast (fibroblast, and human umbilical vein endothelial cell's strain (human umbilical vein cell line NIH/3T3), EA.hy926) migration (in vitro wound healing assay) way is with reference to Rodriguez (2005) 10) research, its entirety is all quoted the reference data of doing this case.Cell kind is entered in the hole of 12 little lattice culture plates (12well plate), every little lattice kind enters 5*10
5individual cell, is positioned over 37 DEG C, 5%CO
2incubator after 24 hours, original cell culture fluid is removed, and utilizes 200 μ l tip to mark the scar of one level.Then add 200 μ l PBS rinses once, then take pictures after adding the cell culture fluid that contains thick extraction thing, then be positioned over 37 DEG C, 5%CO
2incubator after 24 hours, take pictures in same block, utilize image analysing computer software (TScratch version1.0) to analyze photo and do sorting-out in statistics after obtaining data.
2. experimental result
Referring to Figure 14, it carries out in vitro wound healing test experiments result with the fibroblast of mouse: NIH/3T3, and in figure, A, B are control group, do not give tester; C, D are BP2 low dose group, and concentration is 10 μ g/ml; E, F are BP2 high dose group, and concentration is 20 μ g/ml; G, H are BP3 low dose group, and concentration is 10 μ g/ml; I, J are BP3 high dose group, and concentration is 20 μ g/ml.And utilize the area of scratch part in SPOT Advanced computed in software image, carry out statistical analysis after obtaining the area that the 24th hour cell covers scratch, as K in figure.Result shows, gives the low dose group of BP2 and the cell area coverage of high dose group all than the obvious lifting of control group.
Referring to Figure 15, it carries out in vitro wound healing test experiments result with human umbilical vein endothelial cell's strain: EA.hy926, and in figure, A, B are control group, do not give tester; C, D are BP2 low dose group, and concentration is 10 μ g/ml; E, F are BP2 high dose group, and concentration is 20 μ g/ml; G, H are BP3 low dose group, and concentration is 10 μ g/ml; I, J are BP3 high dose group, and concentration is 20 μ g/ml.And utilize the area of scratch part in SPOT Advanced computed in software image, carry out statistical analysis after obtaining the area that the 24th hour cell covers scratch.Result shows, giving BP2, BP3 low dose group all has remarkable increase under comparing with the cell area coverage of control group.
B. live body wound healing promoting activity analysis
1. wound healing test:
A. laboratory animal is prepared: the public Mus of Wistar rat that laboratory animal is 400-450g, raise in the animal housing of Zhongshan University's marine resources shop.Photoperiod maintains 12 hours dark of illumination in 12 hours, and rat can freely be taken drinking-water and food.And with the temperature and humidity of Air-condition system control feeding environment, make ambient temperature remain on 23 DEG C.
B. burn and scald wound is manufactured: the activity analysis of this live body wound healing promoting is with reference to Huang (2008) 11), its entirety is all quoted the reference data of doing this case.By after rat random packet, use animal to shave a mao machine back defeathering with (anesthetic machine: Isotec4, Ohmeda) under 2.5%isoflurane anesthesia, re-use razor and shave except clean.Utilize after alcohol disinfecting, use knife blade rat back to be removed to the holostrome skin of 2 centimeters of diameters, treatment group gives medicine immediately according to experimental design, and injured group is not done any treatment, and rear every the rat of having performed the operation is independently raised.
C. give natural goods to wound: same time natural goods (this natural goods is that the blank base that Corallium Japonicum Kishinouye slightly extracts thing and provides with applicant mixes use, and wherein the detailed composition of this blank base is referring to following) was provided was evenly spread on wound every day.Before each treatment, first wound is cleaned remaining day before yesterday natural goods cleanly with sterile saline, remove foreign body simultaneously, then after normal saline solution being wiped and done by aseptic swab stick, the even drug of topical application on wound.
D. wound observe with area calculate: according to experimental design day aspire to after burn and scald, rat anesthesia is placed on rephotograph stand, with digital camera (Coolpix P6000, Nikon, Japan) under the same conditions (aperture 7.2, shutter 1/60 second) take a series of photos.Use digital image acquiring systems soft ware (Diagnostic Instruments, Inc., Sterling Heights, MI, USA), analytical photography gained wound photo is to calculate wound area.The data that put wound area each observing time present part, are respectively to show with respect to the percentage ratio mode of the 0th day wound area.Measure rat body weight simultaneously, and observe rat and have or not in outward appearance or behavior obviously difference.
2. experimental result
Referring to Figure 16, it is placed in rat back 10 seconds with 175 DEG C of copper billets, creates respectively 4 and scalds wounds, gives afterwards BP2, the BP3 of each wound 3mg/0.2ml every day, pure cream is made the result for the treatment of.Its result confirms to scald the relation of post burn (day) and wound recovery area (%), and after administration, in 8 days, BP3 is better with the effect of controlling wound area to suppressing inflammation, and BP2 had preferably curative effect after 8 days.
As shown in figure 17, BP2, the BP3 of the another 40 μ g/0.2ml with variable concentrations, thick extraction, pure emulsion are to scalding Wound healing and bone regeneration.The relation that confirms to scald post burn (day) and wound recovery area (%) in figure, finishes since the 2nd day to experiment, and BP3 and BP2 group have the ability of accelerating to treat wound.
C. tissue slice test:
1. test procedure:
A. histopathologic slide and HE dyeing:
Rat is given after human sacrifice in injured rear specified number of days according to experimental design, with 4 DEG C of PBS that contain heparin (0.2U/ml) by aorta perfusion, until vein flows out the not PBS with color.Again with 4% paraformaldehyde (paraformaldehyde) perfusion fixation of 4 DEG C.Finally with scalpel, injured area is carefully taken off, be soaked in 10% formalin fixative and leave under 4 DEG C of environment and fix a couple of days.
Next be fixed tissue and dewater and ooze wax processing, utilize and organize automated processing system that skin histology is dewatered and ooze wax, then with paraffin organization embedding machine, organization embedding is become to paraffin mass.Then carry out after tissue slice with paraffin slicing machine, use haematoxylin-Yihong staining to carry out tissue section strain.After completing, with mounting rubber seal sheet, then the sample slide completing is placed in to observation by light microscope, takes and record section result in conjunction with digital image acquiring system.
Histopathologic slide analyze operating process with reference to Bayat (2005) 12) method, its entirety is all quoted the reference data of doing this case.Under the 400X visual field, in the skin corium of each skin histology sample, choose at random 20 blocks, and further carry out quantitatively for leukocyte numbers such as neutrophils, macrophage and lymph corpuscles.In addition in order to assess compromised skin recovery, choose at random 20 points so each at 3 positions such as epidermis, corium and striped muscle of each skin histology sample respectively, measure these 3 layers points other thickness.Analyzing about above these histopathologic slides, is all to be operated by unwitting experimenter that animal is divided into groups.
B. immunohistochemistry staining method:
Skin histology is fixed after two hours after with 4% paraformaldehyde, be transposed to overnight in 30% sucrose solution of 4 DEG C after.For lowering the difference in immunohistochemistry, this case adopts the certainly previous research of amendment (Sung et al., 2003; Chen et al., 2008) method 13,14) operates.Therefore each group of skin histology is embedded in same piece of tissue.The complete piece of tissue of embedding is used freezing microtome at-30 DEG C, to carry out tissue slice, and every slice thickness is 5 μ m.Frozen section sample after dry 1 hour, is placed in to 4% paraformaldehyde solution 10 minutes under room temperature.Under room temperature, react after 1 hour with the lowlenthal serum (4%) of PBS dilution again, be covered in respectively the PBS (containing 0.01%Triton X-100 and 2% lowlenthal serum) that contains antibody and be placed at 4 DEG C overnight.Then, at room temperature react 1 hour with the secondary monoclonal antibody with green fluorescence (Alexa Fluor488-conjugated secondary monoclonal antibody) or with secondary polyclonal antibody (rhodamine-conjugated secondary polyclonal antibody) the covering section of red fluorescence respectively.After finishing, secondary polyclonal antibody utilizes Leica DM-6000B fluorescence microscope (Leica, Wetzlar, Germany) observe, and use SPOT Xplorer digital image acquiring system (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) pick-up image.In the time observing green fluorescence, set the laser ripple of fluorescence microscope and be longer than 488nm; And while observing red fluorescence image, be that to set laser wavelength be 568nm.When each acquisition fluoroscopic image, all use 2.5 times of same time of exposure of object lens immobile phase, it is identical that image size all keeps under every kind of experiment condition.Unwitting observer uses this cover software (National Institutes of Health, Bethesda, MD, USA) of Image J to calculate for each group of immunocompetence not being apprised of under experiment content.
2. result of the test:
Figure 18 utilizes HE dyeing (5X) to inquire into the impact of thick extraction thing for the section of rat burn and scald skin histopathology, in Figure 18, A is that in control group, Figure 18, B is that in burn and scald group, Figure 18, C is that in BP2 group, Figure 18, D is BP3 group, gives natural goods concentration and is all 3mg/0.2ml.Can be seen by the HE result of taking pictures, with respect to the control group without injured (A), burn and scald group (B) all has with the epithelial layer for the treatment of group (C, D) phenomenon thickening, especially burn and scald group (B) is thick more many than treatment group (C), with respect to burn and scald group, the epidermal area surface for the treatment of group (C) is comparatively level and smooth.In burn and scald group and treatment group, all can find granulation tissue, show that burn and scald can cause that dermal tissue is impaired, infiltrate (inflammatory cell infitration) with luring inflammatory cells into, and the degree that treatment group infiltrates be lower than burn and scald group.
Figure 19 utilizes HE dyeing (5X) to inquire into the impact of thick extraction thing for the section of rat burn and scald skin histopathology, in Figure 19, A is that in control group, Figure 19, B is that in burn and scald group, Figure 19, C is that in BP2 group, Figure 19, D is that in BP3 group, Figure 19, E, for thick extraction thing group, gives natural goods concentration and is all 40 μ g/0.2ml.Can be seen by the HE result of taking pictures, with respect to the control group without injured (A), burn and scald group (B) all has and bites in a large number neutral leukocyte generation with the epithelial layer for the treatment of group (E), in the epithelial layer for the treatment of group (C, D), bite neutral leukocyte less, and treatment group (C) there is hair follicle regeneration.
According to above result, show that BP2 and BP3 are for scalding wound and have the situation of acceleration recovery, and low concentration 40 μ g/0.2m time seem there is better curative effect when at 3mg/0.2ml.
Test case 4: soft and soggy Corallium Japonicum Kishinouye effective ingredient embryo skin care products effect test
1. skin quality detector carries out skin quality and improves monitoring:
Carry out the assessment of skin quality effect with the multi-functional skin quality detector of Aramo-TS, the inboard arm subregion of 10 testees is smeared test sample (blank base every day, Corallium Japonicum Kishinouye cream: BP2 (200 μ g/ml) cream, BP3 (200 μ g/ml) cream, Corallium Japonicum Kishinouye slightly extracts (200 μ g/ml)) cream)), and in testing weekly, continue to carry out 6 weeks, result is smeared the difference of rear and background value with pairedStudent ' sT-Test statistical analysis, the effective ingredient of blank base and soft and soggy Corallium Japonicum Kishinouye effective ingredient embryo skin care products arranges as following table 2, the difference of its empty base and Corallium Japonicum Kishinouye cream is only having or not for Corallium Japonicum Kishinouye extract.
2. result of the test
Result is as shown in Figure 20 and Figure 21, and after experimenter uses the embryo skin care products 4 weeks that adds soft and soggy Corallium Japonicum Kishinouye effective ingredient, the testee that has clear improvement is in moisturizing degree and the elasticity of skin, especially best with BP2 cream effect of Corallium Japonicum Kishinouye.
Above-described embodiment and accompanying drawing non-limiting product form of the present invention and style, suitable variation or modification that any person of an ordinary skill in the technical field does it, all should be considered as not departing from patent category of the present invention.
Claims (19)
- For skin-whitening, moisturizing, improve the Corallium Japonicum Kishinouye extract of elasticity, anti-inflammatory and wound healing, it is characterized in that, obtained by the following step:A. with dry soft and soggy Corallium Japonicum Kishinouye (Briareum excavatum) sample of an organic liquid mixture extraction, obtain just extract of organic layer, the C1-C4 alcohols that wherein this organic liquid mixture comprises equal proportion and the chloride alkanes of C1-C4;B. distribute just extract of this organic layer of extraction with water and rudimentary esters, obtain a rudimentary esters and slightly extract thing, this rudimentary esters slightly extracts thing and contains a Corallium Japonicum Kishinouye active component of 5～50%.
- 2. Corallium Japonicum Kishinouye extract as claimed in claim 1, it is characterized in that: this rudimentary esters of b. step slightly extracts thing further with tubing string chromatographic analysis purification in addition, it rushes extract punching with one and carries this rudimentary esters and slightly extract thing, with one rush extract, wherein this rushes rushing of extract and puies forward gradient and provided by one first mixed solvent of n-hexane/ethyl acetate and one second mixed solvent of ethyl acetate/methanol.
- 3. Corallium Japonicum Kishinouye extract as claimed in claim 1, is characterized in that: the C1-C4 alcohols of a. step is methanol.
- 4. Corallium Japonicum Kishinouye extract as claimed in claim 1, is characterized in that: the chloride alkanes of the C1-C4 of a. step is dichloromethane.
- 5. Corallium Japonicum Kishinouye extract as claimed in claim 1, is characterized in that: the rudimentary esters of b. step is ethyl acetate.
- 6. Corallium Japonicum Kishinouye extract as claimed in claim 2, is characterized in that: in this first mixed solvent, the solvent gradient of normal hexane is 0%-99%.
- 7. Corallium Japonicum Kishinouye extract as claimed in claim 2, is characterized in that: in this second mixed solvent, the solvent gradient ratio of ethyl acetate is 0%-90%.
- 8. Corallium Japonicum Kishinouye extract as claimed in claim 6, it is characterized in that: this Corallium Japonicum Kishinouye active component slightly extracts thing by this rudimentary esters and further rushed and carried and obtain by this first mixed solvent, wherein rushing of this first mixed solvent put forward gradient and is: normal hexane: the division (fraction) that ethyl acetate is 90:10 to 70:30.
- 9. Corallium Japonicum Kishinouye extract as claimed in claim 8, is characterized in that: it is 5-30% that this Corallium Japonicum Kishinouye active component slightly extracts thing proportion in this rudimentary esters after punching is carried.
- 10. Corallium Japonicum Kishinouye extract as claimed in claim 1, it is characterized in that: this Corallium Japonicum Kishinouye active component slightly extracts thing by this rudimentary esters and further rushed and carried and obtain by this first mixed solvent, wherein rushing of this first mixed solvent put forward gradient and is: normal hexane: the division (fraction) that ethyl acetate is 60:40 to 50:50.
- 11. Corallium Japonicum Kishinouye extracts as claimed in claim 10, is characterized in that: it is 20-50% that this Corallium Japonicum Kishinouye active component slightly extracts thing proportion in this rudimentary esters after punching is carried.
- The extracting process of 12. 1 kinds of Corallium Japonicum Kishinouye extracts as claimed in claim 1, comprises:A. with dry soft and soggy Corallium Japonicum Kishinouye (Briareum excavatum) sample of an organic liquid mixture extraction, obtain just extract of organic layer, the C1-C4 alcohols that wherein this organic liquid mixture comprises equal proportion and the chloride alkanes of C1-C4;B. distribute just extract of this organic layer of extraction with water and rudimentary esters, obtain a rudimentary esters and slightly extract thing, this rudimentary esters slightly extracts thing and contains a Corallium Japonicum Kishinouye active component of 5～50%.
- 13. extracting process as claimed in claim 12, it is characterized in that: this rudimentary esters of b. step slightly extracts thing further with tubing string chromatographic analysis purification in addition, it rushes extract punching with one and carries this rudimentary esters and slightly extract thing, with one rush extract, wherein this rushes rushing of extract and puies forward gradient and provided by one first mixed solvent of n-hexane/ethyl acetate and one second mixed solvent of ethyl acetate/methanol.
- The purposes of 14. 1 kinds of Corallium Japonicum Kishinouye extracts, it is applied to Corallium Japonicum Kishinouye extract as claimed in claim 1 the skin of one live body, so that skin-whitening.
- The purposes of 15. 1 kinds of Corallium Japonicum Kishinouye extracts, it is applied to Corallium Japonicum Kishinouye extract as claimed in claim 1 the skin of one live body, so that skin moisture-keeping.
- The purposes of 16. 1 kinds of Corallium Japonicum Kishinouye extracts, it is applied to Corallium Japonicum Kishinouye extract as claimed in claim 1 the skin of one live body, to improve skin elasticity.
- The purposes of 17. 1 kinds of Corallium Japonicum Kishinouye extracts, it uses Corallium Japonicum Kishinouye extract as claimed in claim 1, with anti-inflammatory.
- The purposes of 18. 1 kinds of Corallium Japonicum Kishinouye extracts, it uses Corallium Japonicum Kishinouye extract as claimed in claim 1, to impel wound healing.
- 19. 1 kinds of skin care productss, it comprises Corallium Japonicum Kishinouye extract as described in claim 1～11 any one, it is characterized in that: the weight ratio of this Corallium Japonicum Kishinouye extract tool 0.00001～10%.
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| TW102111780A TWI487539B (en) | 2013-04-01 | 2013-04-01 | Coral extract for skin whitening, moisture retention, elasticity improvement and occlusion of wounds and thereof skincare product |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101688453B1 (en) * | 2016-06-20 | 2016-12-22 | 박지영 | Cosmetic composition for skin whitening, reducing skin pore size and secreting sebum |
| CN109010225A (en) * | 2018-08-22 | 2018-12-18 | 海南热带海洋学院 | A kind of coral reef peel off pack and preparation method thereof |
| CN111317748A (en) * | 2018-12-14 | 2020-06-23 | 中山大学 | Coral compound extract, composition containing the same and preparation method thereof |
| CN115671148A (en) * | 2021-07-28 | 2023-02-03 | 台湾中山大学 | Sea fungus extract and application thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4153127A1 (en) * | 2020-05-19 | 2023-03-29 | L'oreal | Anti-acne compositions |
| FR3112283A1 (en) * | 2020-07-07 | 2022-01-14 | L'oreal | Anti-acne compositions |
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| US20090258091A1 (en) * | 2008-04-09 | 2009-10-15 | Marine Bio Co., Ltd. | Compositions and methods for treating neurodegenerative diseases |
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- 2014-01-22 US US14/160,727 patent/US20140294745A1/en not_active Abandoned
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| CN101057877A (en) * | 2006-04-21 | 2007-10-24 | 中国医学科学院药物研究所 | Sacandra glabra extracting method, extraction and its medicinal composition and application |
| TW200823220A (en) * | 2006-11-28 | 2008-06-01 | Nat Museum Of Marine Biology & Amp Aquarium | Novel briarane-related diterpenoid compound, its preparing and application method |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101688453B1 (en) * | 2016-06-20 | 2016-12-22 | 박지영 | Cosmetic composition for skin whitening, reducing skin pore size and secreting sebum |
| CN109010225A (en) * | 2018-08-22 | 2018-12-18 | 海南热带海洋学院 | A kind of coral reef peel off pack and preparation method thereof |
| CN111317748A (en) * | 2018-12-14 | 2020-06-23 | 中山大学 | Coral compound extract, composition containing the same and preparation method thereof |
| US11628191B2 (en) | 2018-12-14 | 2023-04-18 | National Sun Yat-Sen University | Coral composite extract, composition including the same and method of producing the same |
| US11738055B2 (en) | 2018-12-14 | 2023-08-29 | National Sun Yat-Sen University | Coral composite extract, composition including the same and method of producing the same |
| US11963985B2 (en) | 2018-12-14 | 2024-04-23 | National Sun Yat-Sen University | Coral composite extract, composition including the same and method of producing the same |
| CN115671148A (en) * | 2021-07-28 | 2023-02-03 | 台湾中山大学 | Sea fungus extract and application thereof |
| CN115671148B (en) * | 2021-07-28 | 2023-10-03 | 台湾中山大学 | Auricularia auricula extract and application thereof |
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| US20140294745A1 (en) | 2014-10-02 |
| TWI487539B (en) | 2015-06-11 |
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